The past fifteen years witnessed the transformation of animal cell culture from an exploratory protein production method to a mature manufacturing process. This technological transformation was aided by significant advances in bioprocessing research and development. As the technology becomes more mature the pressure on driving the production cost down is mounting.
Mammalian cells are capable of utilizing different proportions of glucose and glutamine and metabolize them differently under different culture conditions. Early work by Zielke et. al. (J. Cell. Physiol., 95, 41-48 (1978) showed that glutamine becomes a predominant source of energy at low glucose concentrations. The metabolism of glucose is also greatly affected by glucose concentration in the medium (W.-S. Hu et al., Dev. Biol. Standard., 66, 279-290 (1987); W.-S. Hu et al. in: Large Scale Mammalian Cell Culture Technology, Ed. A. S. Lubineicky, pp. 451-481, Marcel Dekker, Inc., New York, N.Y. (1990)). At high glucose levels, the consumption rate of glucose is higher, although most of the glucose consumed (80-90%) is converted to lactate; at low glucose concentrations, the consumption rate for glucose is lower and a larger proportion is completely oxidized to CO.sub.2.
This tendency of mammalian cells to metabolize glucose to lactate greatly restricts the cell concentration achievable in the bioreactor. Accumulation of lactate in the medium is detrimental to cell growth and is one of the factors that limits the maximum cell concentration that can be achieved in batch culture. In a typical batch cell culture, growth is inhibited after lactate concentration in the culture reaches approximately 30-50 mM and/or ammonia concentration reaches 3-5 mM. As a result, cell concentration in a batch culture is typically in the range of 1 to 3.times.10.sup.6 cells/ml.
Accumulation of lactate can be reduced by changing from a batch mode into a simple continuous operation, thereby continuously removing the spent culture medium containing lactate and cells. Eventually the concentrations of cells, medium components, and metabolites all stabilize, reaching a steady state. However, using the same medium and resulting steady state cell concentration is, at most, about the same level as that reached in batch mode. Cell concentrations usually cannot be much improved by merely increasing the nutrient concentration in the feed, because an increase in nutrient levels in the feed is generally followed by increased lactate and ammonia production and accumulation, thereby preventing the desired increase in cell concentration.
Perfusion cultures have also been used in attempts to achieve high cell concentrations in a bioreactor, and productivity is significantly higher than in conventional batch cultures. However, the large through-put requirement makes medium preparation for perfusion cultures a daunting task. In a perfusion culture, medium is perfused through the reactor at a high rate while cells are retained or recycled back into the reactor by sedimentation, centrifugation or filtration. Up to ten reactor volumes of medium is perfused through the bioreactor in a day. The major function of perfusing such a large volume of medium is primarily to remove the metabolites, mainly lactate, from the culture fluid. The large demand for medium, the high flow rates, and the multitudinous technical problems associated with cell retention devices make the perfusion cultures expensive, labor-intensive, and prone to technical difficulties.
Attempts have been made to increase cell concentration in fed-batch cultures by controlling glucose and/or glutamine concentrations in an effort to reduce the accumulation of inhibitory metabolites. See, e.g., Kurokowa et al., Biotechnol. Bioeng., 44, 95-103(1994); G.-S. Oh et al., J. Fermentation Bioeng., 81, 319-336 (1996); A. Sanfeliu et al., Biotechnol. Prog., 12, 209-216 (1996); W. Zhou et al., Biotechnol. Bioeng., 46, 579-587 (1995). However, in most cases, growth rates and maximum cell concentration are still limited (e.g., J. Ljunggren et al., Biotechnol. Bioeng. 44, 808-818 (1994)), and cell viability in fed-batch cultures is usually rather low.
Cell concentration in large-scale mammalian cell cultures has thus been limited by the heretofore unavoidable accumulation of inhibitory metabolites in the culture medium, and methods to address the problem have been directed to removing inhibitory metabolites, such as lactate and ammonia, from the culture. A different approach, comprising a method for culturing cells that avoids excess production and accumulation of toxic metabolites such as lactate in the first place, would represent a far better solution to this long-standing problem.